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Investigating glial glycogen utilization for ion homeostasis in the brain and its relevance to epileptogenesis: electrophysiology and pharmacology in awake behaving mice

Periodic Reporting for period 1 - GLION (Investigating glial glycogen utilization for ion homeostasis in the brain and its relevance to epileptogenesis: electrophysiology and pharmacology in awake behaving mice)

Reporting period: 2016-03-01 to 2018-02-28

The brain is one of the most energetically expensive organs in the body and energetic constraints limit brain information processing. Utilization of cerebral glycogen (polysaccharide of glucose, the sole form of glucose storage in the brain) within the glycolytic pathway has recently be shown to be critical for basic as well as higher brain functions, including neuronal excitability, sleep-wake cycle and cognitive abilities such as learning and memory. However, the exact reasons for why glycogenolysis and glycolysis are so essential to brain function remain unknown.
The identification of the mechanisms underlying functional metabolism in the brain holds great potential to understand the relevance of energetic requirements to both physiological and pathological conditions. Indeed, impairments in brain metabolism often precede aberrant neuronal excitability and cognitive decline, which together represent significant social burdens and, as such, demand for effective therapeutic interventions.
The overall objective of the present project is to increase our comprehension about the role of glycogen in neuronal activity, from basic cellular processes to the overall function of the organ. In particular, glycogen in the brain is confined to glial astrocytes and is implicated in the maintenance of homeostasis through the control of the levels of neuroactive compounds. Among these, potassium is a key regulator of neuronal excitability and involvement of glycogenolysis in potassium buffering is an established function for brain astrocytes. Furthermore, astrocytic glycogen is implicated in neurotransmitter homeostasis, both excitatory glutamate and inhibitory GABA through specific metabolic pathways.
The conclusions of the action are supportive of the importance of glycogen and glycolysis in governing local as well as global brain activity in response to sensory stimulation, and in particular during conditions of increased noradrenergic tone. The latter finding indicates that increased glycolytic metabolism rises during a reorienting behavioral response, which is known to be associated with memory formation.
The central hypothesis of the present project about the role of glycogen in energy metabolism and potassium/neurotransmitter homeostasis has been tested using state-of-the-art in vivo optical imaging coupled to potassium-sensitive electrodes, electrophysiology and biochemical techniques.
The work carried out towards the objectives included the study of the regional differences in brain metabolism of glucose in awake and anesthetized animals as well as in animals whose cerebral glycogenolysis was inhibited pharmacologically. Animals underwent intracisternal injections of either isotope-labeled or fluorescent glucose in the brain cerebrospinal fluid under different conditions. Focused-beam microwave irradiation was employed to avoid post-mortem metabolism and brain regions were micro-dissected and analyzed using biochemical techniques, high-performance liquid chromatography and gas chromatography/mass spectrometry. The dynamics of extracellular potassium and signatures of epileptic-like activity was studied in the cerebral cortex of awake and anesthetized animals under different conditions (e.g. resting, sensory stimulation, startle). Fluorescent probes (including fluorescent glucose analogues and genetically-encoded calcium indicators) were used in head-restrained animals in combination with two-photon microscopy or wide-field transcranial imaging and electrophysiological recordings. The role of glycogen breakdown in astrocytes was assessed by pharmacological inhibition of the enzyme degrading glycogen.
Project outcomes included the association between brain state and tissue glycogen content as well as significant correlations between glycogen and neurotransmitter levels. Glycogen was also found to be one of the target of behavioral responses rather than simple (i.e. not associated with behavior) sensory stimulation, together with potassium, an index of neuronal activity, and lactate, a metabolic product of glycogenolysis and glycolysis. Part of the results obtained are published in international journals as peer-reviewed articles. So far, two articles are already published and two more are in preparation. Three collaborative papers (related to the project, but not included as main scientific output) are published as well. In addition, results have been presented at international conferences, and also as outreach activities for the general public organized by local institutions (i.e. outside academia).
The project did not yield specific intellectual property nor technological improvements. However, benefits for the research community, achieved through presentations of results at local meetings and international conferences, include the important consideration for the field that brain glycogen status (tissue level and homogeneity of the molecule) is extremely important tough very challenging to measure quantitatively and technical advancements can be undoubtedly identified as necessary future developments.
The present project confirms and extends recent findings supporting an essential role for glycogen as a dynamic storage of glucose molecules in the brain. In particular, glycogen actively participates in brain potassium and neurotransmitter homeostasis, as evidenced by concentration changes with brain state (anesthesia/sleep-wake), sensory and/or behavioral stimulation. Data shows that glycogen utilization is significantly correlated with the levels of important metabolites including glutamine, glutamate and GABA, which constitute the major neurotransmitter system in the brain. Glycolytic activity (i.e. production of lactate) is also found to be state-dependent, but uncorrelated to the well-known changes in respiratory quotient taking place during state transitions. These observations indicate that other compounds are involved. Experiments that were not anticipated in the present project but suggested by the outcomes of the project, are presently ongoing (preliminary results obtained during the project) in the laboratory, focusing on the involvement of ketone bodies and fatty-acids oxidation in cortical state changes and epileptogenesis. Together with previous work, these studies advance the field and provide novel concepts to the study of functional brain energy metabolism.
The project dramatically improved the technical skills and the networking opportunity of the researcher. In addition, the project set the basis for proficient collaborations between the different parties involved in the project. For instance, the researcher and the host institution are actively collaborating to put the results of the present project into new grant applications, with the involvement of national and international institutions and scientists.
Besides the impact in planning and executing further scientific activities, the project provided information of interest for the society. During public engagement, results were discussed to emphasize the importance of diet (relevant to substrate availability for energy metabolism) and life style (relevant to a proper balance between sleep and wakefulness as well as stress/fatigue) to brain health. The latter has direct societal implications, as a correct diet and life style might help reducing the long-term drug use that is common in many pathologies.
Example of experimental measurements showing correlation between brain state and glycogen.